Studies on the structure, function, and assembly of the Escherichia coli (E. coli) 30S ribosomal subunit were revolutionized when it was discovered that a mixture of the natural 30S ribosomal proteins (TP30) could be reconstituted with 16S ribosomal RNA (rRNA) into functional 30S subunits (Traub & Nomura, 1968). Subsequently, the individual protein components (S1-S21) of the 30S subunit were identified, purified, and characterized (Hardy et al., 1969; Nomura et al., 1969; Traut et al., 1969; Kaltschmidt & Wittmann, 1971). Individually purified ribosomal proteins added as a mixture could also be reconstituted with 16S rRNA into functional 30S subunits (Mizushima & Nomura, 1970; Held et al., 1974). The reconstituted 30S subunits were shown to have the same sedimentation behavior and protein composition as natural 30S subunits and were shown to function in tRNA binding and polyphenylalanine (polyPhe) synthesis. Taken together, these experiments demonstrated that 30S subunits are capable of self-assembly, and that all of the information required for in vitro assembly is contained within these molecular components.
While the ability to reconstitute E. coli 30S subunits in vitro from purified natural components allowed detailed investigation of the structure, function, and assembly of the 30S subunit, isolation of sufficient amounts of highly purified, functional small subunit ribosomal proteins from isolated subunits was difficult, laborious, and costly. In particular, it was difficult to exclude cross-contamination between ribosomal proteins in large-scale purification. Culver and Noller (1999) cloned and overexpressed a complete set of recombinant small subunit ribosomal proteins and developed an ordered assembly protocol that allowed efficient reconstitution of 30S subunits using the purified recombinant proteins. In particular, Culver and Noller (1999) found that reconstitution of 30S subunits with recombinant proteins was less efficient than those reconstituted using a complete mixture of natural ribosomal proteins (TP30) but more efficient than 30S subunit reconstitution using proteins individually purified from ribosomal subunits. The molecular composition and sedimentation properties of the recombinant 30S subunits were similar to those of natural 30S subunits and those reconstituted with TP30, and the recombinant 30S subunits were active, as measured by in vitro assays for tRNAPhe binding and polyPhe synthesis (Culver and Noller, 1999).
Based on in vitro reconstitution of functional 30S subunits using a mixture of total proteins isolated from the 30S subunit (TP30), individually purified natural (Held et al., 1973) or recombinant (Culver and Noller, 1999) small subunit proteins, and 16S rRNA, the association of 16S rRNA and various ribosomal proteins during 30S subunit formation was determined (FIG. 1A) (Mizushima and Nomura, 1970; Held et al., 1974). Some of the small subunit proteins, the primary binding proteins (1°; FIG. 1A), can bind independently and specifically to naked 16S rRNA. The secondary binding proteins (2°; FIG. 1A) require the prior association of at least one primary binding protein before they are able to interact appropriately with the growing RNP. Finally, the tertiary binding proteins (3°; FIG. 1A) bind once primary and secondary binding proteins have associated to complete the cooperative assembly of functional 30S subunits. Studies on the dynamics of in vitro 30S subunit assembly have shown that different regions of 16S rRNA undergo assembly at different rates (Powers et al., 1993), likely influenced by a combination of protein-dependent RNA conformational changes and rates of association of different proteins.
In vitro 30S subunit assembly is characterized by slow kinetics and is dependent upon optimal temperature and ionic conditions. At low temperatures, where E. coli growth is uncompromised, reconstitution stalls and a particle which sediments at 21S is formed (FIG. 1B) (Traub and Nomura, 1968). The 21S reconstitution intermediate (RI) contains a subset of the small subunit ribosomal proteins, corresponding to the primary and secondary proteins, and 16S rRNA (I; FIG. 1B) (Held and Nomura, 1973). A temperature-dependent conformational change is required to convert RI to an assembly competent intermediate, RI* which sediments at 26S (II; FIG. 1B). Once ΔRI* is formed, it is competent for assembly of the tertiary binding proteins (III; FIG. 1B). An intermediate similar to RI has been observed in vivo (Guthrie et al., 1969; Nashimoto et al., 1971; Nierhaus et al., 1973; Lindahl, 1975; Alix et al., 1993), strongly suggesting that in vitro and in vivo 30S subunit assembly follows similar paths.
Nevertheless, the observed in vitro and in vivo intermediates and the characteristics of in vitro 30S subunit reconstitution suggest that factors that normally assist 30S subunit assembly in vivo are lacking in the in vitro systems described above. Maki et al. (2002) identified components of the DnaK chaperone system that facilitated assembly of a functional 30S subunit from recombinant small subunit ribosomal proteins and 16S rRNA. However, Alix et al. (2003) disputed whether DnaK had a role in in vitro formation of functional 30S subunits.
Thus, it needs to be determined whether the DnaK chaperone system facilitates assembly of a functional 30S subunit.